Targeted cell free nucleic acid analysis

ABSTRACT

Methods of isolating cell free RNA from individual&#39;s bodily fluid and reliably obtain cell free RNA data are presented, preferably by use of high-stability portions and/or use of targeted small amplicons on the cell free RNA.

This application is a divisional of U.S. patent application Ser. No.16/759,577, filed on Apr. 27, 2020, which claims priority to U.S.Provisional Application No. 62/582,619, filed on Nov. 7, 2017, which isincorporated by reference in its entirety herein.

FIELD OF THE INVENTION

The field of the invention is diagnostic methods in cancer therapy,especially as it relates to diagnosis, prognosis, and treatment ofcancer using liquid biopsy.

BACKGROUND OF THE INVENTION

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

Genetic abnormalities or abnormal expression of genes are frequentlyassociated with prognosis of many diseases including inflammatorydiseases, autoimmune diseases, metabolic diseases, and various types ofcancer. Most typically, the genetic abnormalities or abnormal expressionof genes are determined by examining tissues and/or analyzing omics dataof the tissues obtained by excisional or incisional biopsy. Whileexcisional or incisional biopsy directly provide relevant tissue andcells from which omics data can be obtained, such procedures are oftennot desirable and may not be performed frequently due to theinvasiveness and difficulties to gain access to the target tissue.

More recently, liquid biopsies using cell free (or free circulating) DNAand/or RNA populations in peripheral blood have become an at leastconceptually simple method for the analysis of genetic abnormalitiesassociated with prognosis of cancer. For example, U.S. Pat. No.9,422,592 discloses the measurement of cell free RNA (cfRNA) offormulpeptide receptor gene (FPR1) and its association with thepatient's risk for having lung cancer or non-small cell lung cancer(NSCLC). However, as the quantity of cell free RNA of specific gene ofinterest (e.g., a gene encoding tumor-specific epitope) is generallymarginal in a limited volume of bodily fluid, and as cell free RNA tendsto degrade rapidly, omics data on some cell free RNA may not be reliablyobtained and analyzed. So far, most efforts have been made to solve theproblem by increasing the overall yield of cell free RNAs during thepurification step and by reducing the amount of RNA degradation usingRNAase inhibitor or chemicals of similar functions. Yet, such approachmay not be equally effective for all nucleic acids, and will not improvedetection where RNA is already degraded in vivo.

Therefore, even though various methods of purification of RNA withreduced RNA degradation are known, individual gene-specific approach forreliably and stably obtaining and analyzing cell free RNA has beenlargely unexplored. Thus, there remains a need for improved methods forreliably and stably obtaining and analyzing cell free RNA.

SUMMARY OF THE INVENTION

The inventive subject matter is directed to various compositions andmethods of isolating cell free RNA and reliably obtaining cell free RNAdata at a desirable signal to noise ratio, even when the RNA ispartially degraded. Thus, in one aspect of the inventive subject matter,the inventors contemplate a method of obtaining cell free RNA data. Inthis method, a sample containing cell free RNA from an individual isobtained. Preferably, the sample is a bodily fluid of the individual.Then, a high-stability portion of the cell free RNA is amplified toobtain the cell free RNA data, which includes at least one of RNAsequence data and the RNA expression level.

Preferably, the cell free RNA is derived from at least one of thefollowing: a cancer-related gene, a cancer-specific gene, a DNA-repairgene, a neoepitope, and a gene not associated with a disease. In someembodiments, the neoepitope is tumor-specific and individual-specific.In other embodiments, the cell free RNA is a small noncoding RNA.

In some embodiments, the high-stability portion can be identified bylength or structure of the portion, a location within the cell free RNA,an interaction of the high-stability portion with a protein, anempirical analysis of portions of the cell free RNA, and/or by in silicomodeling of the cell free RNA. The structure of the portion may compriseat least one of a hairpin structure, a loop structure, a pseudoknotstructure, and a bulge structure. The location may be within 200 basepairs from 5′-end of the cell free RNA. In some embodiments, the insilico modeling presents the predicted secondary structure in vivo or invitro.

In some embodiments, amplifying a high-stability portion of the cellfree RNA comprises amplifying fragments of the high-stability portion indifferent lengths. In such embodiments, it is preferred that thefragments are amplified in different lengths using a plurality ofdistinct 5′-primers or distinct 3′-primers.

In some embodiments, the RNA sequence data are selected from the groupconsisting of mRNA sequence data and splice variant data, and/or the RNAexpression level data are selected from the group consisting of aquantity of RNA transcript and a quantity of a small noncoding RNA.

Another aspect of the inventive subject matter includes a method ofisolating cell free RNA. In this method, a sample containing cell freeRNA from an individual is obtained and subsequently contacted with asynthetic nucleic acid. Preferably, the synthetic nucleic acid isconfigured to bind to at least a portion of 5′-portion of the cell freeRNA and form a cell free RNA-synthetic nucleic acid complex. Mosttypically, the sample is a bodily fluid of the individual. The so formedcell free RNA-synthetic nucleic acid complex is isolated and the methodoptionally can be continued with analyzing the cell free RNA associatedwith the synthetic nucleic acid or cell free RNA dissociated from thesynthetic nucleic acid.

In some embodiments, the cell free RNA is derived from at least one ofthe following: a cancer-related gene, a cancer-specific gene, aDNA-repair gene, a neoepitope, and a gene not associated with a disease.Preferably, the neoepitope is tumor-specific and individual-specific. Inother embodiments, cell free RNA is a small noncoding RNA.

In some embodiments, the synthetic nucleic acid is a double-strandedDNA, and the cell free RNA-synthetic nucleic acid complex is a DNA-RNAtriplex. In other embodiments, the synthetic nucleic acid is asingle-stranded DNA, and cell free RNA-synthetic nucleic acid complex isa DNA-RNA hybrid double helix.

In some embodiments, the synthetic nucleic acid is immobilized via atleast one of a nanoparticle, a magnetic bead, a glass bead, a biotinbead, and a quantum dot, and/or is immobilized on the solid carrier viaa covalent bonding to a surface of the solid carrier.

In some embodiments, the portion of the 5′-portion of the cell free RNAis within 500 or 200 base pairs from 5′-end of the cell free RNA. Inother embodiments, the portion of the 5′-portion of the cell free RNA iswithin 150 or 120 base pairs from 5′-end of the cell free RNA.

In some embodiments, the step of isolating comprises separating theRNA-synthetic nucleic acid complex by at least one of a change inmolecular weight and a conformational change. In other embodiments, thesynthetic nucleic acid is labeled with a tag, and the step of isolatingcomprises separating the RNA-synthetic nucleic acid complex using thetag.

Additionally, the method may further comprise a step of amplifying thecell free RNA from the isolated cell free RNA-synthetic nucleic acidcomplex to obtain the cell free RNA data, wherein the cell free RNA datacomprises at least one of RNA sequence data and RNA expression leveldata. In such embodiments, it is preferred that the RNA sequence dataare selected from the group consisting of mRNA sequence data and splicevariant data and/or the RNA expression level data are selected from thegroup consisting of a quantity of RNA transcript and a quantity of asmall noncoding RNA.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments.

DETAILED DESCRIPTION

The inventors discovered that cell free RNA omics data can be reliablyobtained by identifying and amplifying a high-stability portion of thecell free RNA. Such amplified high-stability portion of the cell freeRNA can be further analyzed to obtain cell free RNA sequence data and/orRNA expression level data. Viewed from a different perspective, theinventors discovered that primers for reliably amplifying a portion ofcell free RNA can be designed and generated based on the identifiedhigh-stability portion of the cell free RNA. The inventors furtherdiscovered that the high-stability portion of the cell free RNA can beidentified based on the length, structure, location, or any interactionof the portion with other molecules, which can be determinedexperimentally, empirically, or by in silico modeling. The inventorscontemplate that such targeted amplification of cell free RNA sequenceswill improve the quality and reliability of the omics data by capturingcell free RNAs that may be relatively unstable and prone to degradationin vitro. Of course, it should be noted that RNA data are notnecessarily limited to RNA, but may be represented as DNA sequence datawhere the analysis of the RNA included a step of reverse transcription.

As used herein, the term “tumor” refers to, and is interchangeably usedwith one or more cancer cells, cancer tissues, malignant tumor cells, ormalignant tumor tissue, that can be placed or found in one or moreanatomical locations in a human body. It should be noted that the term“patient” as used herein includes both individuals that are diagnosedwith a condition (e.g., cancer) as well as individuals undergoingexamination and/or testing for the purpose of detecting or identifying acondition. As used herein, the term “bind” refers to, and can beinterchangeably used with a term “recognize” and/or “detect”, aninteraction between two molecules with a high affinity with a K_(D) ofequal or less than 10⁻⁶ M, or equal or less than 10⁻⁷ M. As used herein,the term “provide” or “providing” refers to and includes any acts ofmanufacturing, generating, placing, enabling to use, or making ready touse.

Cell-Free RNA

The inventors contemplate that an individual with a medical conditionthat is related to alteration of genes or gene expressions may beidentified via liquid biopsy of the individual's bodily fluid or samplesderived from the bodily fluid that may include cell free RNA. Forexample, treatment of a cancer patient with one or more cancerimmunotherapy can trigger release of cell free RNA to the patient'sbodily fluid, thus increase the quantity of specific types of the cellfree RNA. As used herein, the individual's bodily fluid includes, but isnot limited to, blood, serum, plasma, mucus, cerebrospinal fluid,ascites fluid, saliva, and urine of the individual. The individual'sbodily fluid may be fresh or preserved/frozen.

As used herein, the cell free RNA may include any types of RNA that arecirculating in the bodily fluid of an individual without being enclosedin a cell body or a nucleus. Most typically, the source of the cell freeRNA is the cell directly or indirectly affected by the medical condition(e.g., cancer, etc.) or treatment to the medical condition (e.g., cancerimmunotherapy). Thus, in one example, the source of the cell free RNAcan be preferably a cancer cell. However, it is also contemplated thatthe source of the cell free RNA is the immune cell (e.g., NK cells, Tcells, macrophages, etc.). Thus, where the medical condition is a tumor(or a cancer), the cell free RNA can be circulating tumor RNA (ctRNA)and/or cell free RNA (cfRNA, circulating nucleic acids that do notderive from a tumor). While not wishing to be bound by a particulartheory, it is contemplated that the release of cell free RNA originatedfrom the tumor cell can be increased when the tumor cell interact withthe immune cell or when the tumor cells undergo cell death (e.g.,necrosis, apoptosis, autophagy, etc.). Thus, in some embodiments, thecell free RNA may be enclosed in a vesicular structure (e.g., viaexosomal release of cytoplasmic substances) so that it can be protectedfrom RNase activity in some type of bodily fluid. Yet, it is alsocontemplated that in other embodiments, the cell free RNA is a naked RNAwithout being enclosed in any membranous structure, but may bestabilized via interaction with non-nucleotide molecules (e.g., any RNAbinding proteins, etc.).

Therefore, in addition to quantification of any known cell free RNA, itis contemplated that the methods presented herein will also includequantification of any cell free RNA and/or specific fractions thereof todetermine the presence of absence of a medical condition or theprognosis of the medical condition in the patient. Where specificfractions are quantified, it should be appreciated that such fractionsmay be particularly relevant to the specific disease. For example,especially suitable RNA fractions include those representing tumorassociated genes and/or neoepitopes specific to a tumor in the patient(tumor-specific and/or patient-specific. Alternatively, or additionally,circulating RNA encoding DNA repair genes are also deemed suitable. Aswill be readily appreciated, such additional measurements may be used asa baseline and/or as an indicator of treatment efficacy. Examples forsuitable methods are disclosed in co-pending U.S. provisionalapplications 62/504,149, filed May 10, 2007, 62/473,273, filed Mar. 17,2017, and 62/500,497 filed May 3, 2017, all incorporated by referenceherein.

It is contemplated that the cell free RNA can be any type of RNA whichcan be released from either cancer cells or immune cell. Thus, the cellfree RNA may include mRNA, tRNA, microRNA, small interfering RNA, longnon-coding RNA (lncRNA). Most typically, the cell free RNA is a fulllength or a fragment of mRNA (e.g., at least 70% of full-length, atleast 50% of full length, at least 30% of full length, etc.) encodingone or more cancer-related proteins, inflammation-related proteins,cancer neoepitope (preferably patient-specific and tumor-specific). Forexample, the cell free mRNA are derived from the cancer related geneincluding, but not limited to, ABL1, ABL2, ACTB, ACVR1B, AKT1, AKT2,AKT3, ALK, AMER11, APC, AR, ARAF, ARFRP1, ARID1A, ARID1B, ASXL1, ATF1,ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BCL2, BCL2L1,BCL2L2, BCL6, BCOR, BCORL1, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD4,BRIP1, BTG1, BTK, EMSY, CARD11, CBFB, CBL, CCND1, CCND2, CCND3, CCNE1,CD274, CD79A, CD79B, CDC73, CDH1, CDK12, CDK4, CDK6, CDK8, CDKN1A,CDKN1B, CDKN2A, CDKN2B, CDKN2C, CEA, CEBPA, CHD2, CHD4, CHEK1, CHEK2,CIC, CREBBP, CRKL, CRLF2, CSF1R, CTCF, CTLA4, CTNNA1, CTNNB1, CUL3,CYLD, DAXX, DDR2, DEPTOR, DICER1, DNMT3A, DOT1L, EGFR, EP300, EPCAM,EPHA3, EPHAS, EPHA7, EPHB1, ERBB2, ERBB3, ERBB4, EREG, ERG, ERRFI1,ESR1, EWSR1, EZH2, FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG,FANCL, FAS, FAT1, FBXW7, FGF10, FGF14, FGF19, FGF23, FGF3, FGF4, FGF6,FGFR1, FGFR2, FGFR3, FGFR4, FH, FLCN, FLI1, FLT1, FLT3, FLT4, FOLH1,FOXL2, FOXP1, FRS2, FUBP1, GABRA6, GATA1, GATA2, GATA3, GATA4, GATA6,GID4, GLI1, GNA11, GNA13, GNAQ, GNAS, GPR124, GRIN2A, GRM3, GSK3B,H3F3A, HAVCR2, HGF, HMGB1, HMGB2, HMGB3, HNF1A, HRAS, HSD3B1, HSP9OAA1,IDH1, IDH2, IDO, IGF1R, IGF2, IKBKE, IKZF1, IL7R, INHBA, INPP4B, IRF2,IRF4, IRS2, JAK1, JAK2, JAK3, JUN, MYST3, KDMSA, KDMSC, KDM6A, KDR,KEAP, KEL, KIT, KLHL6, KLK3, MLL, MLL2, MLL3, KRAS, LAG3, LMO1, LRP1B,LYN, LZTR1, MAGI2, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MCL1, MDM2, MDM4,MED12, MEF2B, MEN1, MET, MITF, MLH1, MPL, MRE11A, MSH2, MSH6, MTOR,MUC1, MUTYH, MYC, MYCL, MYCN, MYD88, MYH, NF1, NF2, NFE2L2, NFKB1A,NKX2-1, NOTCH1, NOTCH2, NOTCH3, NPM1, NRAS, NSD1, NTRK1, NTRK2, NTRK3,NUP93, PAK3, PALB2, PARK2, PAX3, PAX, PBRM1, PDGFRA, PDCD1, PDCD1LG2,PDGFRB, PDK1, PGR, PIK3C2B, PIK3CA, PIK3CB, PIK3CG, PIK3R1, PIK3R2,PLCG2, PMS2, POLD1, POLE, PPP2R1A, PREX2, PRKAR1A, PRKC1, PRKDC, PRSS8,PTCH1, PTEN, PTPN11, QK1, RAC1, RAD50, RAD51, RAF 1, RANBP1, RARA, RB1,RBM10, RET, RICTOR, RIT1, RNF43, ROS1, RPTOR, RUNX1, RUNX1T1, SDHA,SDHB, SDHC, SDHD, SETD2, SF3B1, SLIT2, SMAD2, SMAD3, SMAD4, SMARCA4,SMARCB1, SMO, SNCAIP, SOCS1, SOX10, SOX2, SOX9, SPEN, SPOP, SPTA1, SRC,STAG2, STAT3, STAT4, STK11, SUFU, SYK, T (BRACHYURY), TAF1, TBX3, TERC,TERT, TET2, TGFRB2, TNFAIP3, TNFRSF14, TOP1, TOP2A, TP53, TSC1, TSC2,TSHR, U2AF1, VEGFA, VHL, WISP3, WT1, XPO1, ZBTB2, ZNF217, ZNF703, CD26,CD49F, CD44, CD49F, CD13, CD15, CD29, CD151, CD138, CD166, CD133, CD45,CD90, CD24, CD44, CD38, CD47, CD96, CD 45, CD90, ABCB5, ABCG2, ALCAM,ALPHA-FETOPROTEIN, DLL1, DLL3, DLL4, ENDOGLIN, GJA1, OVASTACIN, AMACR,NESTIN, STRO-1 , MICL, ALDH, BMI-1, GLI-2, CXCR1, CXCR2, CX3CR1, CX3CL1,CXCR4, PON1, TROP1, LGR5, MSI-1, C-MAF, TNFRSF7, TNFRSF16, SOX2,PODOPLANIN, L1CAM, HIF-2 ALPHA, TFRC, ERCC1, TUBB3, TOP1, TOP2A, TOP2B,ENOX2, TYMP, TYMS, FOLR1, GPNMB, PAPPA, GART, EBNA1, EBNA2, LMP1, BAGE,BAGE2, BCMA, C10ORF54, CD4, CD8, CD19, CD20, CD25, CD30, CD33, CD80,CD86, CD123, CD276, CCL1, CCL2, CCL3, CCL4, CCLS, CCL7, CCL8, CCL11,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22,CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCR1, CCR2, CCR3, CCR4, CCRS,CCR6, CCR7, CCR8, CCR9, CCR10, CXCL1, CXCL2, CXCL3, CXCLS, CXCL6, CXCL9,CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, CXCL17, CXCR3, CXCRS,CXCR6, CTAG1B, CTAG2, CTAG1, CTAG4, CTAGS, CTAG6, CTAG9, CAGE1, GAGE1,GAGE2A, GAGE2B, GAGE2C, GAGE2D, GAGE2E, GAGE4, GAGE10, GAGE12D, GAGE12F,GAGE12J, GAGE13, HHLA2, ICOSLG, LAG1, MAGEA10, MAGEA12, MAGEA1, MAGEA2,MAGEA3, MAGEA4, MAGEA4, MAGEAS, MAGEA6, MAGEA7, MAGEA8, MAGEA9, MAGEB1,MAGEB2, MAGEB3, MAGEB4, MAGEB6, MAGEB10, MAGEB16, MAGEB18, MAGEC1,MAGEC2, MAGEC3, MAGED1, MAGED2, MAGED4, MAGED4B, MAGEE1, MAGEE2, MAGEF1,MAGEH1, MAGEL2, NCR3LG1, SLAMF7, SPAG1, SPAG4, SPAG5,SPAG6, SPAG7,SPAG8, SPAG9, SPAG11A, SPAG11B, SPAG16, SPAG17, VTCN1, XAGE1D, XAGE2,XAGE3, XAGE5, XCL1, XCL2, and XCR1. Of course, it should be appreciatedthat the above genes may be wild type or mutated versions, includingmissense or nonsense mutations, insertions, deletions, fusions, and/ortranslocations, all of which may or may not cause formation offull-length mRNA.

For another example, the cell free mRNA are those encoding a full lengthor a fragment of inflammation-related proteins, including, but notlimited to, HMGB1, HMGB2, HMGB3, MUC1, VWF, MMP, CRP, PBEF1, TNF-α,TGF-β, PDGFA, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, FGF, G-CSF, GM-CSF, IFN-γ,IP-10, MCP-1, PDGF, and hTERT, and in yet another example, the cell freemRNA encoded a full length or a fragment of HMGB1.

For still another example, the cell free mRNA are those encodingDNA-repair proteins, including, but not limited to, DNA glycosylase,APE1, XRCC1, PNKP, Tdpl, APTX, DNA polymerase β, FEN1, DNA polymerase δor ε, PCNA-RFC, PARP, MutSa (MSH2-MSH6), MutSβ (MSH2-MSH3), MutLα(MLH1-PMS2), MutLβ (MLH1-PMS2), MutLγ (MLH1-MLH3), Exo1, PCNA-RFC,XPC-Rad23B-CEN2, UV-DDB (DDB1-XPE), CSA, CSB, TFIIH, XPB, XPD, XPA, RPA,XPG, ERCC1-XPF, DNA polymerase δ or ε, Mrell-Rad50-Nbs1, CtIP, RPA,Rad51, Rad52, BRCA1, BRCA2, Exol, BLM-TopIIIα, GEN1-Yen1, Slx1- Slx4,Mus81/Eme1, and Ku70-Ku80, DNA-PKc, XRCC4-DNA ligase IV, XLF.

The cell free mRNA may be present in a plurality of isoforms (e.g.,splicing variants, etc.) that may be associated with different celltypes and/or location. Preferably, different isoforms of mRNA may be ahallmark of specific tissues (e.g., brain, intestine, adipose tissue,muscle, etc.), or may be a hallmark of cancer (e.g., different isoformis present in the cancer cell compared to corresponding normal cell, orthe ratio of different isoforms is different in the cancer cell comparedto corresponding normal cell, etc.). For example, mRNA encoding HMGB1are present in 18 different alternative splicing variants and 2unspliced forms. Those isoforms are expected to express in differenttissues/locations of the patient's body (e.g., isoform A is specific toprostate, isoform B is specific to brain, isoform C is specific tospleen, etc.). Thus, in these embodiments, identifying the isoforms ofcell free mRNA in the patient's bodily fluid can provide information onthe origin (e.g., cell type, tissue type, etc.) of the cell free mRNA.

The inventors contemplate that the quantities and/or isoforms (orsubtypes) or regulatory noncoding RNA (e.g., microRNA, small interferingRNA, long non-coding RNA (lncRNA), etc.) can vary and fluctuate bypresence of a tumor or immune response against the tumor. Withoutwishing to be bound by any specific theory, varied expression ofregulatory noncoding RNA in a cancer patient's bodily fluid may due togenetic modification of the cancer cell (e.g., deletion, translocationof parts of a chromosome, etc.), and/or inflammation at the cancertissue by immune system (e.g., regulation of miR-29 family by activationof interferon signaling and/or virus infection, etc.). Thus, in someembodiments, the cell free RNA can be a regulatory noncoding RNA thatmodulates expression (e.g., downregulates, silences, etc.) of mRNAencoding a cancer-related protein or an inflammation-related protein(e.g., HMGB1, HMGB2, HMGB3, MUC1, VWF, MMP, CRP, PBEF1, TNF-α, TGF-β,PDGFA, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-17, Eotaxin, FGF, G-CSF, GM-CSF, IFN-γ, IP-10,MCP-1, PDGF, hTERT, etc.).

It is also contemplated that some cell free regulatory noncoding RNA maybe present in a plurality of isoforms or members (e.g., members ofmiR-29 family, etc.) that may be associated with different cell typesand/or location. Preferably, different isoforms or members of regulatorynoncoding RNA may be a hallmark of specific tissues (e.g., brain,intestine, adipose tissue, muscle, etc.), or may be a hallmark of cancer(e.g., different isoform is present in the cancer cell compared tocorresponding normal cell, or the ratio of different isoforms isdifferent in the cancer cell compared to corresponding normal cell,etc.). For example, higher expression level of miR-155 in the bodilyfluid can be associated with the presence of breast tumor, and thereduced expression level of miR-155 can be associated with reduced sizeof breast tumor. Thus, in these embodiments, identifying the isoforms ofcell free regulatory noncoding RNA in the patient's bodily fluid canprovide information on the origin (e.g., cell type, tissue type, etc.)of the cell free regulatory noncoding RNA.

Isolation of Cell Free RNA

Any suitable methods to isolate cell free RNA are contemplated. Mosttypically, cell free RNA is isolated from a bodily fluid (e.g., wholeblood, serum, etc.) or any sample that may contain cell free RNA of theindividual that is processed under conditions that stabilize cell freemRNA. In some embodiments, the bodily fluid of the patient can beobtained from a patient before and after the cancer immunotherapy. Whileit may vary depending on the type of cancer immunotherapy and/or thetype of cancer, the bodily fluid of the patient can be obtained at least24 hours, at least 3 days, at least 7 days after the cancerimmunotherapy. For more accurate comparison, the bodily fluid from thepatient before the cancer immunotherapy can be obtained less than 1hour, less than 6 hours before, less than 24 hours before, less than aweek before the beginning of the cancer immunotherapy. In addition, aplurality of samples of the bodily fluid of the patient can be obtainedduring a period before and/or after the cancer immunotherapy (e.g., oncea day after 24 hours for 7 days, etc.).

Additionally or alternatively, the bodily fluid of a healthy individualcan be obtained to compare the quantity and/or subtype expression ofcell free RNA. As used herein, a healthy individual refers an individualwithout a tumor. Preferably, the healthy individual can be chosen amonggroup of people shares characteristics with the patient (e.g., age,gender, ethnicity, diet, living environment, family history, etc.).

In more detail, suitable tissue sources include whole blood, which ispreferably provided as plasma or serum. Alternatively, it should benoted that various other bodily fluids are also deemed appropriate solong as cell free RNA is present in such fluids. Appropriate fluidsinclude saliva, ascites fluid, spinal fluid, urine, etc., which may befresh or preserved/frozen. For example, for the analyses presentedherein, specimens were accepted as 10 ml of whole blood drawn intocell-free RNA BCT® tubes or cell-free DNA BCT® tubes containing RNAstabilizers, respectively. Advantageously, cell free RNA is stable inwhole blood in the cell-free RNA BCT tubes for seven days while cellfree RNA is stable in whole blood in the cell-free DNA BCT Tubes forfourteen days, allowing time for shipping of patient samples fromworld-wide locations without the degradation of cell free RNA. Moreover,it is generally preferred that the cell free RNA is isolated using RNAstabilization agents that will not or substantially not (e.g., equal orless than 1%, or equal or less than 0.1%, or equal or less than 0.01%,or equal or less than 0.001%) lyse blood cells. Viewed from a differentperspective, the RNA stabilization reagents will not lead to asubstantial increase (e.g., increase in total RNA no more than 10%, orno more than 5%, or no more than 2%, or no more than 1%) in RNAquantities in serum or plasma after the reagents are combined withblood. Likewise, these reagents will also preserve physical integrity ofthe cells in the blood to reduce or even eliminate release of cellularRNA found in blood cell. Such preservation may be in form of collectedblood that may or may not have been separated. In less preferredaspects, contemplated reagents will stabilize cell free RNA in acollected tissue other than blood for at 2 days, more preferably atleast 5 days, and most preferably at least 7 days. Of course, it shouldbe recognized that numerous other collection modalities are also deemedappropriate, and that the cell free RNA can be at least partiallypurified or adsorbed to a solid phase to so increase stability prior tofurther processing.

As will be readily appreciated, fractionation of plasma and extractionof cell free RNA can be done in numerous manners. In one exemplarypreferred aspect, whole blood in 10 mL tubes is centrifuged tofractionate plasma at 1600 rcf for 20 minutes. The so obtained plasma isthen separated and centrifuged at 16,000 rcf for 10 minutes to removecell debris. Of course, various alternative centrifugal protocols arealso deemed suitable so long as the centrifugation will not lead tosubstantial cell lysis (e.g., lysis of no more than 1%, or no more than0.1%, or no more than 0.01%, or no more than 0.001% of all cells). Cellfree RNA is extracted from 2 mL of plasma using Qiagen reagents. Theextraction protocol was designed to remove potential contaminating bloodcells, other impurities, and maintain stability of the nucleic acidsduring the extraction. All nucleic acids were kept in bar-coded matrixstorage tubes, with DNA stored at −4° C. and RNA stored at −80° C. orreverse-transcribed to cDNA that is then stored at −4° C. Notably, soisolated cell free RNA can be frozen prior to further processing.

Alternatively, the inventors contemplate that some cell free RNAs can betargeted and isolated from other cell free RNAs by providing a syntheticor recombinant nucleic acid that can bind to the cell free RNA. Anysuitable synthetic or recombinant nucleic acids that can bind andstabilize the cell free RNA are contemplated. For example, the syntheticor recombinant nucleic acid can be a double-stranded DNA that hascomplementary sequences with the cell free RNA. As used herein, thecomplementary sequences includes any sequences that can stably bind tothe cell free RNA. Thus, the complementary sequences may includesequences fully complementary to the cell free RNA, at least 95%complementary, at least 90% complementary, at least 80% complementary,or at least 70% complementary to the cell free RNA. In this example, thecell free RNA may bind near or within the major groove of thedouble-stranded DNA to form a RNA-DNA triplex, at least partially. Foranother example, the synthetic or recombinant nucleic acid can be asingle-stranded DNA that has complementary sequences with the cell freeRNA, which can, together, form a DNA-RNA hybrid double helix, at leastpartially. Without wishing to be bound by any specific theory, theinventors contemplate that so formed RNA-DNA triplex or DNA-RNA hybriddouble helix may protect the cell free RNA from RNase-mediateddegradation or any other mechanism by which single-stranded RNA are morevulnerable to be degraded.

Preferably, the synthetic or recombinant nucleic acid includes nucleicacid sequences complementary to at least 20%, preferably at least 30%,more preferably at least 50% of length of the cell free RNA. Inaddition, while any portion of the cell free RNA may bind to thesynthetic or recombinant nucleic acid, it is generally preferred thatthe synthetic or recombinant nucleic acid may include nucleic acidsequences complementary to at least a portion of first one fifth (⅕) of5′-portion of the cell free RNA, first one fourth (¼) of 5′-portion ofthe cell free RNA, or first one third (⅓) of 5′-portion of the cell freeRNA. For example, where the expected length of the cell free RNA is 150base pairs (bps), the synthetic or recombinant nucleic acid includes mayinclude nucleic acid sequences complementary to the 30 bps at 5′-end ofcell free RNA (e.g., base pair number 1-30 from 5′-end, base pair number10-40 from 5′-end, base pair number 20-50 from 5′-end, etc.) having atotal length of 150 bps.

In a further preferred embodiment, the synthetic or recombinant nucleicacid may be immobilized on a solid carrier so that the cell free RNAbound to the synthetic or recombinant nucleic acid can be immobilized aswell. Any suitable solid carrier can be used including, but not limitedto, any planar substrate, a chip, a column, a bead, a dipstick-typeformat. Preferably, the synthetic or recombinant nucleic acid can beimmobilized by at least one end (5′-end or 3′-end) of the synthetic orrecombinant nucleic acid. Alternatively, the synthetic or recombinantnucleic acid may be immobilized on a solid carrier via any portion ofthe synthetic or recombinant nucleic acid (e.g., any nucleotide ornucleic acid in between the 5′-end and 3′-end).

In some embodiments, the synthetic or recombinant nucleic acid can beimmobilized on the solid carrier by covalent bonding with the substrateon the carrier. For example, where the substrate is coated with achemical having an aldehyde, the 5′-end of the synthetic or recombinantnucleic acid can form a covalent bonding with the aldehyde group viaSchiff base bond. In other embodiments, the synthetic or recombinantnucleic acid can be coupled with a particle via which the synthetic orrecombinant nucleic acid are immobilized on the solid carrier. Anysuitable particles that can immobilize the nucleic acid include, but notlimited to, a nanoparticle (e.g., a metal nanoparticle, etc.), amagnetic bead, a glass bead, a biotin bead, a quantum dot, or any othersuitable materials. For example, a double-stranded synthetic DNA can becoupled with a magnetic bead at its 5-end, and immobilized on thesurface via magnetic force applied from outside of the solid carrier orfrom the substrate on the surface of the solid carrier. For otherexample, a single-stranded synthetic DNA can be coupled with magneticbeads at each of its 5′-end and 3′-end such that the 5′-end and 3′-endof the single-stranded synthetic DNA can be immobilized in a differentparts of the solid carrier (e.g., across the diameter of a capillarycolumn, etc.).

In those embodiments where the synthetic or recombinant nucleic acid areimmobilized on the solid carrier, the inventors contemplate that asample (e.g., serum, blood, etc.) of an individual containing cell freeRNA can be contacted with the synthetic or recombinant nucleic acid onthe solid carrier so that the some cell free RNA having a complementarysequence with the synthetic or recombinant nucleic acid can be bound andimmobilized on the solid carrier. Then, the unbound cell free RNA orother substances can be washed away to isolate the cell free RNA ofinterest from other nucleic acids in the sample. Thus, the inventorsfurther contemplate that the solid carrier can include immobilizedsynthetic or recombinant nucleic acids having different complementarysequences targeting different cell free RNAs in an array such that aplurality of different cell free RNAs can be isolated from a singlereaction (contact) of the sample with the solid carrier.

Additionally and alternatively, the synthetic or recombinant nucleicacid may be coupled with a nanoparticle or a bead as described above,but may not be immobilized on the solid carrier surface. In thisembodiment, the synthetic or recombinant nucleic acid coupled with ananoparticle or a bead as described can be free floating in a liquidbuffer in a container (e.g., a column, a dish, a capillary, etc.). Thesample can be contacted with the synthetic or recombinant nucleic acidsin the buffer to form a triplex or DNA-RNA hybrid double helix, whichcan then be isolated from the other non-bound cell free RNAs or othersubstances in the sample using the nanoparticle or a bead as a tag(e.g., pull down the magnetic bead-associated nucleic acids usingmagnetic force, etc.).

The inventors further contemplate that the synthetic or recombinantnucleic acid bound to the cell free RNA should have higher molecularweight than other unbound synthetic or recombinant nucleic acid.Alternatively or additionally, the conformations of the synthetic orrecombinant nucleic acid may be changed due to the binding to the cellfree RNA. Thus, in some embodiments, the synthetic or recombinantnucleic acid bound to cell free RNA can be separated from the otherunbound synthetic or recombinant nucleic acid by molecular weight-basedseparation (e.g., gel-electrophoresis, capillary-electrophoresis, etc.)or any other separation method that separates out molecules of differentconformations (e.g., non-denaturing gel electrophoresis, etc.).

Selection of Area and Primers for Amplification

After isolation of the cell free RNA, cell free RNA are then amplifiedto quantify the expression level of the cell free RNA or analyzing itssequences. One of the common challenges in amplifying the isolated cellfree RNA is that the cell free RNA often cannot be effectively amplifieddue to its tendency of degradation such that the quantification of sogenerated amplicon may not provide a reliable reflection of the quantityof cell free RNA present in the sample. The inventors found thatamplicons of the cell free RNA can be more effectively and reliablygenerated using primers that target one or more relatively stable, orhigh-stability portions of the cell free RNA. As used herein, therelatively stable, or high-stability portion of the cell free RNA referssequences in the cell free RNA that tends to be secured or protectedfrom degradation in vitro and/or in vivo for at least 3 hours, 6 hours,12 hours, 24 hours, or 3 days after generation of the cell free RNA orfor at least 5 min, at least 15 min, at least 30 min, at least 1 hour,or at least 6 hours in vitro in a temperature of or higher than 4 degreeCelcius.

Thus, in order to generate amplicons of the portion of cell free RNAsfor reliable analysis, it is preferred to identify one or morehigh-stability portion of the cell free RNA. The inventors contemplatethat a portion with a longer nucleic acid sequence length is less likelyto provide a reliable amount and quality of amplicons as the longernucleic acid may tend to degrade from its 3′-end (e.g., via3′-5′-exonuclease, etc.). Thus, it is further contemplated that aportion with a shorter nucleic acid sequence length can be more stablyand reliably transcribed than a portion with a longer nucleic acidsequence length a high- stability portions of the cell free RNA can beidentified by a length of the portion. Based on this, it is contemplatedthat a high-stability portion of the cell free RNA can be any portionwith a length less than 200 bps, preferably less than 150 bps, morepreferably less than 120 bps, and even more preferably less than 100bps. Thus, in one embodiment, the high-stability portion of the cellfree RNA can be identified based on a length of the portion, and 5′- or3′-primer for amplifying cell free RNA can be selected to amplify nomore than 200 bps, preferably no more than 150 bps, more preferably nomore than 120 bps, and even more preferably no more than 100 bps withinthe cell free RNA. In other word, 5′- or 3′-primer for amplifying cellfree RNA can be selected based on the distance from the 3′- or5′-primer, respectively.

Additionally, or alternatively, the inventors also contemplate that aportion of the cell free RNA at or near 3′-end is less likely to providea reliable amount and quality of amplicons as the 3′-end of the nucleicacid is more vulnerable to the RNase-mediated degradation (e.g., via3′-5′-exonuclease, etc.). Thus, it is further contemplated that aportion at 5′-end or near the 5′-end of the cell free RNA may be morestably and reliably transcribed than a portion at 3′-end or near the3′-end of the cell free RNA. Thus, in one embodiment, the high-stabilityportion of the cell free RNA can be identified based on a location ofthe portion, and 5′- or 3′-primer for amplifying cell free RNA can beselected, designed, and/or generated to amplify no more than 300 bps,preferably no more than 200 bps, or 150bps, or 120 bps, more preferablyno more than 100 bps away from the 5′-end of the cell free RNA. In otherword, 5′- or 3′-primer for amplifying cell free RNA can be selected,designed, and/or generated to amplify a portion of the cell free RNAlocated within 300 bps, preferably 200 bps, more preferably 100 bps fromthe 5′-end of the cell free RNA. Consequently, primers for amplificationcan be located towards the 5′-end of an RNA molecule (e.g., within thefirst 50% or within the first 40% or within the first 30% or within thefirst 20% of bases of the transcript) and selected, designed, and/orgenerated such that the amplicon has a length of equal or less than 200bp, or equal or less than 150 bp, equal or less than 120 bp, equal orless than 100 bp, equal or less than 80 bp, or equal or less than 60 bp.

The inventors also contemplate that a portion of the cell free RNAforming a secondary structure is more likely to provide a reliableamount and quality of amplicons as the secondary structure of RNAprovides structural stability so that it can be less vulnerable todegradation. Thus, in one embodiment, the high-stability portion of thecell free RNA can be identified based on a known secondary structure ofthe portion, which includes, but not limited to a stem-loop structure, ahairpin structure, a loop structure, a pseudoknot structure, and a bulgestructure. Where the secondary structure of the cell free RNA is notexperimentally determined or known, the secondary structure can bepredicted via in silico modeling of the nucleic acid structure.Typically, the in silico modeling of the secondary structure calculatesstructures of the RNA by optimizing the thermodynamic free energy basedon a nearest neighbor energy model. In this embodiment, 5′- or 3′-primerfor amplifying cell free RNA can be selected, designed, and/or generatedto amplify the sequences forming the secondary structure or a fragmentthereof, or a portion of the cell free RNA including sequences formingthe secondary structure and extra nucleic acids next to the secondarystructure. Preferably, the extra nucleic acids amplified with thesequences forming the secondary structure is less than 50 bps,preferably less than 30 bps, more preferably less than 20 bps in either5′- or 3′- of the secondary structure or both.

The inventors further contemplate that a portion of the cell free RNAinteracting and/or binding to a protein (e.g., an RNA-binding proteinexcept RNase) is more likely to provide a reliable amount and quality ofamplicons as the RNA-protein interaction or binding provides structuralstability and protection from RNase activity so that such portion can beless vulnerable to degradation. Thus, in one embodiment, thehigh-stability portion of the cell free RNA can be identified based on aknown protein-interaction sequence in the cell free RNA. Where there isno known protein-interaction sequence in the cell free RNA, aprotein-interaction sequence can be predicted by identifying any similaror consensus sequences in the cell free RNA to known protein-interactionsequence in other RNAs. In this embodiment, 5′- or 3′-primer foramplifying cell free RNA can be selected, designed, and/or generated toamplify the sequences within the protein-interaction sequence or afragment thereof, or a portion of the cell free RNA including theprotein-interaction sequence and extra nucleic acids next to theprotein-interaction sequence. Preferably, the extra nucleic acidsamplified with the protein-interaction sequence is less than 50 bps,preferably less than 30 bps, more preferably less than 20 bps in either5′- or 3′- of the protein-interaction sequence or both.

The inventors further contemplate that a high-stability portion of thecell free RNA that are relatively resistant to common RNA degradationmechanism may tend to survive in samples of other individuals. Thus, inone embodiment, the high-stability portion of the cell free RNA can beidentified based on the empirical study of cell free RNAs detected inother samples from individuals with similar physical and medicalconditions (e.g., age, gender, health status, diseases, diseaseprognosis, etc.), which can be typically conducted by analyzing omicsdata of individuals (e.g., by incrementally producing amplicons along aknown transcript). In this embodiment, 5′- or 3′-primer for amplifyingcell free RNA can be selected, designed, and/or generated to amplifyfrequently found fragment of the cell free RNA, or a fragment no furtherthan 100 bps, preferably less than 50 bps, more preferably less than 20bps from the end of the frequently found fragment.

Thus, in preferred aspect of the inventive subject matter, the inventorscontemplate a method of obtaining cell free RNA data. In this method, asample containing cell free RNA from an individual is obtained.Preferably, the sample is a bodily fluid of the individual. Then, themethod continues with a step of identifying or ascertaining ahigh-stability portion of the cell free RNA. In some embodiments, thehigh-stability portion of the cell free RNA can be identified bydetermining a length of a portion of the cell free RNA that arepreferably less than 200 bps, preferably less than 150 bps, morepreferably less than 120 bps, and even more preferably less than 100bps. In other embodiments, the high-stability portion of the cell freeRNA can be identified by determining a location of a portion of the cellfree RNA that are preferably within 300 bps, preferably 200 bps, morepreferably 100 bps from the 5′-end of the cell free RNA, and/or withinthe first 50% or within the first 40% or within the first 30% or withinthe first 20% of bases of the transcript. In still other embodiments,the high-stability portion of the cell free RNA can be identified bydetermining a secondary structure of a portion of the cell free RNA thatincludes, but not limited to, a stem-loop structure, a hairpinstructure, a loop structure, a pseudoknot structure, and a bulgestructure. In still other embodiments, the high-stability portion of thecell free RNA can be identified by determining a portion of the cellfree RNA interacting and/or binding to a protein. Such portion(s) of thecell free RNA (by length, by location, by secondary structure, bymolecule interaction) can be determined by any suitable experimentalmethods including pull-down assay and/or RNA sequencing.

In still other embodiments, the high-stability portion of the cell freeRNA can be ascertained based on the empirical study of cell free RNAsdetected in other samples from individuals with similar physical andmedical conditions (e.g., age, gender, health status, diseases, diseaseprognosis, etc.), which can be typically conducted by analyzing omicsdata of individuals (e.g., by incrementally producing amplicons along aknown transcript). In this embodiment, 5′- or 3′-primer for amplifyingcell free RNA can be selected, designed, and/or generated to amplifyfrequently found fragment of the cell free RNA, or a fragment no furtherthan 100 bps, preferably less than 50 bps, more preferably less than 20bps from the end of the frequently found fragment. In still otherembodiments, the high-stability portion of the cell free RNA can beascertained by in silico modeling of the nucleic acid structure (forsecondary structure of RNA), and/or by in silico analysis of RNAsequence data of cell free RNA. Additionally and/or alternatively, thehigh-stability portion of the cell free RNA can be ascertained by apriori known and/or implicated high-stability portion of the cell freeRNA that can be that can be obtained from a transcriptomics database.

Once a high-stability portion of the cell free RNA is selected oridentified, the high-stability portion and/or the portion nearby can beamplified using a plurality sets of 5′- and/or 3′-primers to obtain aplurality sets of amplicons to determine the consistency between theabsolute or relative quantities of the amplicons. For example, where thehigh-stability portion of the cell free RNA has a length of 100 bps, a5′-primer for amplifying the high-stability portion can be selected,designed, and/or generated to be complementary to the 5′-end of thehigh-stability portion, and a plurality of 3′-primer can be selected,designed, and/or generated to be complementary to the differentsub-portion of the high-stability portion such that the size of expectedamplicons are, for example, 100 bps, 90 bps, 80 bps, 70 bps, etc. Inanother example, the 3′-primer for amplifying the high-stability portioncan be selected, designed, and/or generated to be complementary to the3′-end of the high-stability portion, and a plurality of 5′-primer canbe selected, designed, and/or generated to be complementary to thedifferent sub-portion of the high-stability portion such that the sizeof expected amplicons are, for example, 100 bps, 90 bps, 80 bps, 70 bps,etc. In still another example, a plurality of 5′- primers and 3′-primerscan be paired to generate amplicons in different sizes. The inventorscontemplate that such approach can reduce the possibility of falsenegative results of presence of the cell free RNA, and may furtherprovide the most desired primer sets to amplify the specific cell freeRNA that can provide amplicons most reliably and stably.

Amplification of Cell Free RNA And Analysis of Cell Free RNA Data

Once the high-stability portion of the cell free RNA is identified anddesired primers to amplify the high-stability portion are designedand/or generated, various cell free RNA data can be obtained fromquantification and/or sequence analysis of the cell free RNA. Withrespect to RNA sequence data, it should be noted that contemplated RNAsequence data includes mRNA sequence data, splice variant data,polyadenylation information, and any other suitable data obtained fromsequencing of RNA molecules. Moreover, it is generally preferred thatthe RNA sequence data can be provided along with a metric for thetranscription strength (e.g., number of transcripts of a damage repairgene per million total transcripts, number of transcripts of a damagerepair gene per total number of transcripts for all damage repair genes,number of transcripts of a damage repair gene per number of transcriptsfor actin or other household gene RNA, etc.), and for the transcriptstability (e.g., a length of poly A tail, etc.). Of course, and as notedabove, the RNA data may be obtained by way of DNA where reversetranscription was employed. Thus, DNA data also represent RNA data.

With respect to the transcription strength (expression level),transcription strength of the cell free RNA can be examined byquantifying the cell free RNA. Quantification of cell free RNA can beperformed in numerous manners, however, expression of analytes ispreferably measured by quantitative real-time RT-PCR of cell free RNAusing primers specific for each gene. For example, amplification can beperformed using an assay in a 10 μL reaction mix containing 2 μL cellfree RNA, primers, and probe. mRNA of α-actin can be used as an internalcontrol for the input level of cell free RNA. A standard curve ofsamples with known concentrations of each analyte was included in eachPCR plate as well as positive and negative controls for each gene. Testsamples were identified by scanning the 2D barcode on the matrix tubescontaining the nucleic acids. Delta Ct (dCT) was calculated from the Ctvalue derived from quantitative PCR (qPCR) amplification for eachanalyte subtracted by the Ct value of actin for each individualpatient's blood sample. Relative expression of patient specimens iscalculated using a standard curve of delta Cts of serial dilutions ofUniversal Human Reference RNA set at a gene expression value of 10 (whenthe delta CTs were plotted against the log concentration of eachanalyte).

Alternatively, where discovery or scanning for new mutations or changesin expression of a particular gene is desired, real time quantitativePCR may be replaced by RNAseq to so cover at least part of a patienttranscriptome. Moreover, it should be appreciated that analysis can beperformed static or over a time course with repeated sampling to obtaina dynamic picture without the need for biopsy of the tumor or ametastasis.

Such obtained RNA sequence data and/or quantification data comprisesomics data of cell free RNA. The sequence data sets may includeunprocessed or processed data sets, and exemplary data sets includethose having BAM format, SAM format, FASTQ format, or FASTA format.However, it is especially preferred that the data sets are provided inBAM format or as BAMBAM diff objects (see e.g., US2012/0059670A1 andUS2012/0066001A1). Moreover, the omics data of the individual can becompared with other individuals or healthy individuals to so obtainpatient and tumor specific information. Further, so obtained omicsinformation can then be processed using pathway analysis (especiallyusing PARADIGM) to identify any impact of any mutations oncancer-related genes, neoepitope genes, or any genes that may be mutatedor differentially expressed in relation to any medical conditions.

Likewise, computational analysis of the sequence data may be performedin numerous manners. In most preferred methods, however, analysis isperformed in silico by location-guided synchronous alignment of cellfree RNA of the patient and a healthy individual as, for example,disclosed in US 2012/0059670A1 and US 2012/0066001A1 using BAM files andBAM servers. Such analysis advantageously reduces false positive dataand significantly reduces demands on memory and computational resources.

With respect to the analysis of cell free RNA of the patient and ahealthy individual, numerous manners are deemed suitable for use hereinso long as such methods will be able to generate a differential sequenceobject. However, it is especially preferred that the differentialsequence object is generated by incremental synchronous alignment of BAMfiles representing genomic sequence information of the cell free DNA/RNAof the patient and a healthy individual. For example, particularlypreferred methods include BAMBAM-based methods as described in US2012/0059670 and US 2012/0066001.

The inventors further contemplate that such obtained RNA sequence dataand/or quantification data can be further used to select and/or generatemore reliable treatment regimen to treat the patient, and further toadminister such treatment regimen (e.g., cell-based therapy,chemotherapy, radiotherapy, vaccination, etc.) to the patient. Forexample, where such obtained RNA sequence data and/or quantificationdata indicates an emergence of a tumor-specific and individual-specificneoepitope (with a specific mutation detected from the sequence data)that are expressed by the tumor cells, the treatment regimen may includea vaccine composition (e.g., a viral vaccine, a bacterial vaccine, ayeast vaccine, etc.) comprising a recombinant nucleic acid encoding theneoepitope. Consequently, the patient can be administered with thevaccine composition in a dose and schedule effective to treat the tumor(e.g., to reduce the tumor size, to increase the immune response againstthe tumor, to increase the survival rate, etc.). In another example,where such obtained RNA sequence data and/or quantification dataindicates overexpression of checkpoint-related genes (e.g., PD-L1,CTLA-4, TIM3, LAG3, etc.), the treatment regimen may include acheckpoint inhibitor (e.g., nivolumab, pembrolizumab, etc.).Consequently, the patient can be administered with the checkpointinhibitor(s) in a dose and schedule effective to treat the tumor (e.g.,to reduce the tumor size, to increase the immune response against thetumor, to increase the survival rate, etc.). In still another example,where such obtained RNA sequence data and/or quantification dataindicates overexpression of specific types of chemokines/cytokines thatinhibits the immune response in the tumor microenvironment, and/orproliferation or (hyper)activation of inhibitory immune cells (e.g.,MDSC, Treg cells, etc.) in the tumor microenvironment, the treatmentregimen may include an antibody or a recombinant molecule having abinding motif to the cytokines/chemokines and/or a cell-based therapy(e.g., genetically modified NK/NKT cells expressing Fas ligand and/orCD40 ligand, etc.) inducing cell death of such inhibitory immune cellsor changing the tumor microenvironment to less immune-suppressive.Conversely, where such obtained RNA sequence data and/or quantificationdata indicates underexpression of specific types of chemokines/cytokinesthat enhance or elicit immune response, the treatment regimen mayinclude immune stimulatory cytokine(s) (e.g., IL-2, IL-8, etc.) and achemokine (e.g., CXCL14, CD40L, CCL2, CCL1, CCL22, CCL17, CXCR3, CXCL9,CXCL10, CXCL11, CXCL14, etc.) or any recombinant molecule including oneor more of such immune stimulatory cytokine(s).

As used herein, the term “administering” refers to both direct andindirect administration of the treatment regimens, drugs, therapiescontemplated herein, where direct administration is typically performedby a health care professional (e.g., physician, nurse, etc.), whileindirect administration typically includes a step of providing or makingthe compounds and compositions available to the health care professionalfor direct administration.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. As used in the description herein and throughoutthe claims that follow, the meaning of “a,” “an,” and “the” includesplural reference unless the context clearly dictates otherwise. Also, asused in the description herein, the meaning of “in” includes “in” and“on” unless the context clearly dictates otherwise. Where thespecification claims refers to at least one of something selected fromthe group consisting of A, B, C . . . and N, the text should beinterpreted as requiring only one element from the group, not A plus N,or B plus N, etc.

What is claimed is:
 1. A method of isolating cell free RNA, comprising:obtaining a sample containing cell free RNA from an individual, whereinthe cell free RNA comprises a high stability portion located within 200base pairs from the 5′-end of the cell free RNA; contacting the samplewith a synthetic nucleic acid, wherein the synthetic nucleic acid isconfigured to bind to at least a portion of 5′-portion of the cell freeRNA and form a cell free RNA-synthetic nucleic acid complex; andisolating the cell free RNA-synthetic nucleic acid complex.
 2. Themethod of claim 1, wherein the sample is a bodily fluid of theindividual.
 3. The method of claim 1, wherein the cell free RNA isderived from at least one of the following: a cancer-related gene, acancer-specific gene, a DNA-repair gene, a neoepitope, and a gene notassociated with a disease.
 4. The method of claim 1, wherein theneoepitope is tumor-specific and individual-specific.
 5. The method ofclaim 1, wherein cell free RNA is a small noncoding RNA.
 6. The methodof claim 1, wherein the synthetic nucleic acid is a double-stranded DNA,and the cell free RNA-synthetic nucleic acid complex is a DNA-RNAtriplex.
 7. The method of claim 1, wherein the synthetic nucleic acid isa single-stranded DNA, and cell free RNA-synthetic nucleic acid complexis a DNA-RNA hybrid double helix.
 8. The method of claim 1, wherein thesynthetic nucleic acid is immobilized on a solid carrier at least by oneend of the synthetic nucleic acid.
 9. The method of claim 8, wherein thesynthetic nucleic acid is immobilized via at least one of ananoparticle, a magnetic bead, a glass bead, a biotin bead, and aquantum dot.
 10. The method of claim 8, wherein the synthetic nucleicacid is immobilized on the solid carrier via a covalent bonding to asurface of the solid carrier.
 11. The method of claim 1, wherein theportion of the 5′-portion of the cell free RNA is within 500 or 200 basepairs from 5′-end of the cell free RNA.
 12. The method of claim 1,wherein the portion of the 5′-portion of the cell free RNA is within 150or 120 base pairs from 5′-end of the cell free RNA.
 13. The method ofclaim 1, wherein the isolating comprises separating the RNA-syntheticnucleic acid complex by at least one of a change in molecular weight anda conformational change.
 14. The method of claim 1, wherein thesynthetic nucleic acid is labeled with a tag, and the isolatingcomprises separating the RNA-synthetic nucleic acid complex using thetag.
 15. The method of claim 1, further comprising amplifying the cellfree RNA from the isolated cell free RNA-synthetic nucleic acid complexto obtain the cell free RNA data, wherein the cell free RNA datacomprises at least one of RNA sequence data and RNA expression leveldata.
 16. The method of claim 15, wherein the RNA sequence data areselected from the group consisting of mRNA sequence data and splicevariant data.
 17. The method of claim 15, wherein the RNA expressionlevel data are selected from the group consisting of a quantity of RNAtranscript and a quantity of a small noncoding RNA.